Graduation Term
Summer 2026
Degree Name
Master of Science (MS)
Department
Department of Chemistry
Committee Chair
Jeremy D. Driskell
Committee Member
Timothy Lash
Committee Member
Jun-Hyun Kim
Abstract
The immobilization of antibodies onto surfaces to act as biorecognition elements is critical to the performance of immunosensors. Ideal immobilization methods will orient antibodies such that the antigen-binding (Fab) regions are readily accessible for antigen capture. Traditional methods of immobilization, such as NHS conjugation and physisorption, yield randomly oriented antibodies, decreasing the sensitivity of immunosensors. Previously, we developed a microbial transglutaminase (mTG)-mediated site-specific conjugation strategy for oriented immobilization of antibodies. Microbial transglutaminase catalyzes the conjugation of a payload to a specific glutamine residue (Q295) on the fragment crystallizable (Fc) region of the antibody, enabling oriented immobilization. This approach significantly improved the antigen-capture activity of immobilized antibodies compared to traditional methods. Here, we aim to further improve this strategy by optimizing the spacer length between the antibody and the surface, a parameter known to influence the accessibility of the Fab regions. To this end, we biotinylated anti-HRP antibodies both randomly and site-specifically, using biotin-antibody polyethylene glycol (PEG) spacers of varying lengths (PEG2, PEG4, PEG12, and PEG24), and immobilized them onto various streptavidin-coated surfaces, including AuNPs, 96-well plates, and Surface Plasmon Resonance (SPR) gold sensors. Antigen capture assays were then conducted to compare the antigen-binding activities of the antibodies. Our results show that whereas PEG length has minimal impact on the antigen-capture abilities of site-specifically conjugated antibodies, longer PEG lengths can significantly enhance the performance of randomly conjugated antibodies. Moreover, the ratio of the activity of site-specific conjugates relative to the random conjugates suggested that the benefit of site-specific conjugation is more pronounced at shorter PEG lengths, where random conjugates underperform. Furthermore, both random and site-specific conjugates showed lower detection limits at longer PEG lengths, with the LOD for random PEG24 being 5 times lower than that for random PEG2, and for site-specific PEG24, 2 times lower than for site-specific PEG2. Finally, quantitation of antibodies on surfaces via Bicinchoninic acid (BCA) assay and SPR showed that either ligand density, orientation, or both could be responsible for observed outcomes.
Access Type
Thesis-Open Access
Recommended Citation
Betangah, Moses Egoh, "Optimization Of Polyethylene Glycol (PEG) Spacer Arm Length for Enhanced Orientation of Surface-Immobilized Antibodies" (2026). Theses and Dissertations. 2381.
https://ir.library.illinoisstate.edu/etd/2381